The present invention relates to a monoclonal antibody which is used for judging a possibility of the onset of bovine leukemia caused by bovine leukemia virus BLV.
The major histocompatibility antigens (MHC antigens) are molecules involved in self-nonself differentiation in the defense mechanism of the living body against infection. They are classified into Class I molecule composed of xcex1 chain and xcex22M, and class II molecule composed of xcex1 chain and xcex2 chain. A groove for trapping an antigen peptide is present on the xcex11 and xcex12 domains, and also on the xcex11 and xcex21 domains. They are featured to have the T cell receptor recognize only a fragmented peptide trapped in the groove, thereby achieve cell death (cellular immunity) by CD8+ cells which have recognized the class I antigens, as well as induce mainly antibody production (humoral immunity) by CD4+ cells which have recognized the class II antigens.
The MHC genes constitute a gene group most full of polymorphism, and the locations of pockets, shapes, sizes, and properties of the peptide trapping grooves are different among haplotypes. It is considered that association conditions of the trapped fragment peptides may vary depending on these differences, which decide immune response and disease sensitivity of each individual. The correlation between the MHC haplotypes and a resistance to a disease (disease insusceptibility) or a possibility of the onset of a disease (disease susceptibility) has been reported, for example, as to human immune deficiency virus (HIV), human T cell leukemia virus (HTLV) and malaria.
As for the bovine MHC (BoLA) class II genes, the existence of DQA, DQB, DRA, DRB, DNA, DOB, DYA, and DYB genes has been estimated so far. DRB3, inter alia, which is one of the three genes (DRB1 to B3) identified on the DRB genetic locus, has been known to encode a functional protein, and the existence of 73 alleles has been revealed so far. However, there is almost no report about correlation between bovine infectious diseases and the bovine MHC (BoLA) haplotypes.
In particular, as to the bovine leukemia virus (BLV), which has the gene pX that regulates virus proliferation in the same manner as the human immunodeficiency virus (HIV) and is a retrovirus most related to HTLV-I, a research group in the United States has reported its relationship with the bovine MHC (BoLA) haplotypes mainly focusing disease resistance; however, its relationship with possibility of onset of the leukemia has not been reported. The ratio of cattle infected by this virus (infection rate in Japan) is 10 to 20%, and 1 to 2% of the infected cattle develops extremely malignant endemic bovine leukemia to die after a long latent period of 10 to 15 years. Therefore, economic loss of stockbreeders caused by the virus is very serious. If a possibility of the onset of cattle after BLV infection can be evaluated by the analysis of bovine MHC (BoLA) haplotypes, it becomes possible to preliminarily select disease resistant cattle for breeding beforehand, and it is expected that extremely safe cattle breeding can be continued.
The inventors of the present invention previously analyzed the structure of DRB gene locus among the bovine MHC (BoLA) class II genes, and reported the structures of DRB3 gene (BoLA-DRB3) and the gene product thereof (Biochem. Biophys. Res. Commun., 209, pp.981-988, 1995). The inventors further studied the function of the gene and found that a portion is present, whose amino acid sequence is distinctly different between cattle developing the leukemia and cattle not developing the disease, in the gene product from the second exon (xcex21 domain) of BoLA-DRB3 showing particularly noticeable polymorphism. They also found that the amino acid substitutions directly correlated with disease susceptibility to BLV and disease resistance. Moreover, they found that, in judging a possibility of the onset of bovine leukemia caused by bovine leukemia virus BLV, a bovine individual, in which an amino acid sequence defined by the amino acid numbers 75to 78 of the xcex21 domain of the bovine MHC Class II DR xcex2 chain is Val-Asp-Thr-Tyr, can be judged to have a possibility of the onset of the leukemia, and they achieved the invention relating to the method (International Publication W098/3680).
A monoclonal antibody (c143 monoclonal antibody) is known to react with a tumor-associated antigen that is excessively expressed in BLV infected cells with progress of pathologic state of bovine leukemia ((a) Aida, Y. et al., Cancer Research, 52, pp.6463-6470, 1992; (b) Aida, Y. et al., Cancer Research, 53, pp.429-437, 1993). The aforementioned publications (a) (p.6469, the left column) and (b) (p.436, the left column) suggested that the tumor-associated antigen recognized by the aforementioned monoclonal antibody is related to an MHC Class II antigen. However, details of the reaction between the monoclonal antibody and the MHC Class II antigens have not been elucidated, and moreover, the structure of the epitope of the aforementioned monoclonal antibody has not been known so far.
When the aforementioned method of judgment (W098/3680) is carried out, it is necessary to collect a sample of a living bovine individual, amplify a desired gene, and then determine a base sequence of exon 2 gene of BoLA-DRB3, or carry out the PCR-RFLP method. The aforementioned publication discloses a primer set useful for the judging method; however, it is troublesome and time-consuming to carry out the aforementioned judging method for numbers of bovine individuals. Accordingly, it has been desired to develop a more simple method for judgment.
Therefore, an object of the present invention is to provide a means of simply and accurately judging a possibility of onset of leukemia in bovine individuals caused by bovine leukemia virus (BLV). More specifically, the object is to provide a means of accurately judging a possibility of onset of leukemia in bovine individuals caused by bovine leukemia virus without necessity of determination of a base sequence of exon 2 of the BoLA-DRB3.
The inventors of the present invention made intensive studies to achieve the foregoing objects. As a result, they found that bovine individuals having a gene, encoding xcex21 domain of the MHC Class II DR xcex2 chain and attributable to a possibility of the onset of bovine leukemia, can be detected with a monoclonal antibody which reacts with a tumor-associated antigen excessively expressed in BLV infecting cells (the c143 monoclonal antibody), and that a possibility of the onset of leukemia can be judged with extremely high accuracy. The present invention was achieved on the basis of the aforementioned findings.
The present invention thus provides c143 monoclonal antibody which is used, for detecting a bovine individual which has a possibility of onset of bovine leukemia; c143 monoclonal antibody which is used for detecting a gene encoding xcex21 domain of the bovine MHC Class II DR xcex2 chain to which a possibility of onset of bovine leukemia is attributable; and c143 monoclonal antibody which is used for detecting a bovine individual which has a gene encoding xcex21 domain of the MHC Class II DR xcex2 chain to which a possibility of onset of bovine leukemia is attributable.
In addition, there are provided a monoclonal antibody which is used for detecting a gene encoding xcex21 domain of the bovine MHC Class II DR xcex2 chain to which a possibility of the onset of bovine leukemia is attributable, wherein the monoclonal antibody has substantially the same reactivity as c143 monoclonal antibody to bovine MHC Class II DR molecule to which a possibility of onset of bovine leukemia is attributable; a monoclonal antibody which is used for detecting a bovine individual having a gene encoding xcex21 domain of the MHC Class II DR xcex2 chain to which a possibility of the onset of bovine leukemia is attributable, wherein the monoclonal antibody has substantially the same reactivity as c143 monoclonal antibody to bovine MHC Class II DR molecule to which a possibility of the onset of bovine leukemia is attributable; and a monoclonal antibody which is used for detecting a bovine individual which has a possibility of onset of bovine leukemia, wherein the monoclonal antibody has substantially the same reactivity as c143 monoclonal antibody to bovine MHC Class II DR molecule to which a possibility of onset of bovine leukemia is attributable.
According to another aspect of the present invention, there is provided an agent for diagnosing a possibility of the onset of bovine leukemia which comprises the aforementioned monoclonal antibody, preferably the aforementioned c143 monoclonal antibody. There are also provided a method for detecting a gene encoding xcex21 domain of bovine MHC Class II DR xcex2 chain to which a possibility of onset of bovine leukemia is attributable by means of c143 monoclonal antibody; a method for detecting a bovine individual having a gene encoding xcex21 domain of MHC Class II DR xcex2 chain to which a possibility of onset of bovine leukemia is attributable by means of c143 monoclonal antibody; and a method for detecting a bovine individual which has a possibility of onset of bovine leukemia by means of c143 monoclonal antibody.
According to still another aspect, there are provided a monoclonal antibody which is capable of detecting a gene encoding xcex21 domain of bovine MHC Class II DR xcex2 chain to which a possibility of onset of bovine leukemia is attributable; a monoclonal antibody which is capable of detecting a bovine individual having a gene encoding xcex21 domain of MHC Class II DR xcex2 chain to which a possibility of the onset of bovine leukemia is attributable; and a monoclonal antibody which capable of detecting a bovine individual having a possibility of onset of bovine leukemia. A preferred example of the monoclonal antibody is c143 monoclonal antibody.